I am trying to cleave a GST tagged protein(about 35 kDa + 26 kDa GST) i have purified the protein but on thrombin cleavage there is very less yeild of my protein with most of it being uncut. I have tried a range of different temperatures, time, buffer conditions but of no use. Can you help? Thanks in advance.
Regards,
Archit

-archit-

Hey,

Its always a problem cleaving the tag off as sometimes the site is buried. People suggest mild denaturation but its always a risk as the protein may loose its activity. I would suggest changing to a small tag like the histidine tag, presence of which is tolerated for activity assays.

Best,
TC

archit on Aug 16 2009, 12:31 AM said:

I am trying to cleave a GST tagged protein(about 35 kDa + 26 kDa GST) i have purified the protein but on thrombin cleavage there is very less yeild of my protein with most of it being uncut. I have tried a range of different temperatures, time, buffer conditions but of no use. Can you help? Thanks in advance.
Regards,
Archit